MOLECULAR AND CELLULAR BIOLOGY, Aug. 2007, p. 5699–5710 Vol. 27, No. 160270-7306/07/$08.00�0 doi:10.1128/MCB.00383-07Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Distinct Endocytic Mechanisms of CD22 (Siglec-2) and Siglec-FReflect Roles in Cell Signaling and Innate Immunity�

Hiroaki Tateno,1 Hongyi Li,1 Melissa J. Schur,3 Nicolai Bovin,2 Paul R. Crocker,4Warren W. Wakarchuk,3 and James C. Paulson1*

Departments of Molecular Biology and Molecular and Experimental Medicine, The Scripps Research Institute, San Diego,California 920371; Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,16/10 ul. Miklukho-Maklaya, 117997 Moscow, Russia2; Institute for Biological Sciences, National Research Council,

Ottawa, Ontario K1A OR6, Canada3; and Division of Cell Biology and Immunology,The Wellcome Trust Biocentre, School of Life Sciences, University of Dundee,

Dow Street, Dundee DD1 5EH, United Kingdom4

Received 2 March 2007/Returned for modification 15 April 2007/Accepted 1 June 2007

Sialic acid-binding immunoglobulin-like lectins (siglecs) are predominately expressed on immune cells. Theyare best known as regulators of cell signaling mediated by cytoplasmic tyrosine motifs and are increasinglyrecognized as receptors for pathogens that bear sialic acid-containing glycans. Most siglec proteins undergoendocytosis, an activity tied to their roles in cell signaling and innate immunity. Here, we investigate theendocytic pathways of two siglec proteins, CD22 (Siglec-2), a regulator of B-cell signaling, and mouse eosin-ophil Siglec-F, a member of the rapidly evolving CD33-related siglec subfamily that are expressed on cells ofthe innate immune system. CD22 exhibits hallmarks of clathrin-mediated endocytosis and traffics to recyclingcompartments, consistent with previous reports demonstrating its localization to clathrin domains. Like CD22,Siglec-F mediates endocytosis of anti-Siglec-F and sialoside ligands, a function requiring intact tyrosine-based motifs. In contrast, however, we find that Siglec-F endocytosis is clathrin and dynamin independent,requires ADP ribosylation factor 6, and traffics to lysosomes. The results suggest that these two siglecproteins have evolved distinct endocytic mechanisms consistent with roles in cell signaling and innateimmunity.

The siglec (sialic-acid binding immunoglobulin [Ig]-like lec-tins) proteins are subset of the Ig superfamily of cell recogni-tion molecules that bind to sialic acid-containing glycans of cellsurface glycoconjugates as ligands (27, 78). Of the 13 humanand 9 murine siglec proteins, 4 are highly conserved in mam-malian species: sialoadhesin (Sn)/Siglec-1, CD22/Siglec-2, my-elin-associated glycoprotein (MAG)/Siglec-4, and Siglec-15.The others comprise a rapidly evolving subfamily homologousto CD33/Siglec-4, known as the CD33-related siglec proteins.With two exceptions (MAG and Siglec-6) the siglec proteinsare predominantly expressed in various white blood cells of theimmune system (25, 43, 78, 85). All contain a N-terminal V-setsugar-binding Ig domain, a variable number of additional C-setIg domains, a transmembrane domain, and a cytoplasmic do-main that typically contains tyrosine-based motifs implicated inregulation of cell signaling and endocytosis.

Many of the siglec proteins contain one or more immunore-ceptor tyrosine-based inhibitory motifs (ITIMs), (I/L/V)XYXX(L/V), suggesting that they play important roles as inhibitoryreceptors of cell signaling (25, 78), as exemplified by CD22,which is well documented as a regulator of B-cell receptor(BCR) signaling. Upon antigen binding to the BCR, the ITIMsof CD22 are quickly tyrosine phosphorylated and recruit pro-

tein tyrosine phosphatase SHP-1, which dephosphorylates theBCR and dampens the B-cell response, setting a threshold forB-cell activation (27, 73).

CD22 is also known to undergo endocytosis following liga-tion by anti-CD22 antibody (38, 64) or high-affinity multiva-lent-sialoside ligands (22). The tyrosine-based ITIMs of CD22also fit the sorting signal YXXØ (where Ø is a hydrophobicresidue) for association with the adaptor complex 2 (AP2),which directs recruitment of receptors into clathrin-coated pits(13). John et al. reported that CD22 associates with the AP50subunit of AP2 through these tyrosine-based motifs and thatthey are required for endocytosis (37). Consistent with thisobservation, CD22 is predominantly localized in clathrin-richdomains (23, 34). Antigen ligation of the BCR results in mo-bilization of the BCR to “activation rafts,” which subsequentlyfuse with clathrin domains prior to endocytosis (69, 70). SinceCD22 is specifically excluded from activation rafts (54), thenegative regulatory effect of CD22 on BCR signaling has beenproposed to occur following its movement to clathrin domains(23), linking the endocytic activity of CD22 to its role in reg-ulation of BCR signaling.

Sn and most CD33-related siglec proteins are expressed oncells of the innate immune system, including monocytes, mac-rophages, neutrophils, eosinophils, and dendritic cells (25, 43,50, 85), focusing investigations into their precise functions onthe known activities of these cells. Like CD22, ligation ofCD33-related siglec proteins (CD33 and Siglec-5, -7, and -9)also induces recruitment of SHP-1 via phosphorylated ITIMs(6, 7, 53, 72). Antibody ligation of the same siglec proteins

* Corresponding author. Mailing address: The Scripps ResearchInstitute, 10550 N. Torrey Pines Rd. MEML71, La Jolla, CA 92037.Phone: (858) 784-9633. Fax: (858) 784-9690. E-mail: jpaulson@scripps.edu.

� Published ahead of print on 11 June 2007.

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initiates their endocytosis, suggesting that endocytic activity isalso a general property of this subfamily (9, 38, 50, 81, 84).Over 20 pathogenic microorganisms express sialic acid-con-taining glycans on their surface (25). Recent demonstration ofthe binding or uptake of several sialylated pathogens, includingNeisseria meningitidis, Trypanosoma cruzi, Campylobacter je-juni, and group B Streptococcus, by Sn and Siglec-5, -7, and -9has suggested various roles of these siglec proteins in the im-mune responses to these organisms (8, 18, 25, 30, 38, 45). Sincemechanisms of pathogen entry and engulfment are increasinglyrecognized to involve the host cells’ endocytic machinery (12),the endocytic functions of the siglec proteins are also relevantto their roles in pathogen recognition and uptake.

In this report, we contrast the mechanisms of endocytosis ofCD22 and a CD33-related siglec, murine Siglec-F, predomi-nately expressed on eosinophils (85). Eosinophils are bestknown for their role in allergic diseases (62). In this regard, arecent report analyzing Siglec-F null mice revealed enhancedeosinophil infiltration and blood eosinophilia in response toallergen challenge, suggesting that Siglec-F is a negative reg-ulator of eosinophil response to allergens (86). Eosinophilsalso contribute to an immune response against foreign patho-gens, including binding, engulfment, and killing of microbes (1,15, 19, 32, 36, 83). While investigating the endocytic activity ofSiglec-F, we observed efficient endocytosis of anti-Siglec anti-body, high-affinity sialoside ligand probes, and Neisseria men-ingitidis bearing sialylated glycans. Like CD22, endocytosiswas dependent on its cytoplasmic ITIM and ITIM-like mo-tifs. Surprisingly, however, Siglec-F exhibited no colocaliza-tion with clathrin, raising doubt that it was endocytosed bya clathrin-mediated mechanism, as proposed for CD22.More detailed investigations showed that while endocytosisof CD22 is indeed mediated by a clathrin-dependent mech-anism and is sorted to early endosome and recycling com-partments, Siglec-F endocytosis is directed to lysosomalcompartments and is mediated by a mechanism that is in-dependent of clathrin and dynamin. The results suggest thatsiglec proteins have evolved different mechanisms of endo-cytosis consistent with roles in regulation of cell signalingand innate immunity.

MATERIALS AND METHODS

Antibodies and reagents. Purified rat anti-Siglec-F (E50-2440), phycoerythrin(PE)-conjugated anti-Siglec-F (E50-2440), fluorescein isothiocyanate (FITC)-conjugated anti-human CD22 (HIB22), PE–Cy5-streptavidin, anti-Syntaxin-8(48), and anti-EEA1 (14 polyclonal antibodies) were purchased from BD Bio-sciences-Pharmingen (San Diego, CA). Anti-hamster LAMP2 (UH3) was pur-chased from the Developmental Studies Hybridoma Bank. Anti-clathrin heavychain (x22) was purchased from Abcam. Affinity-purified sheep anti-Siglec-F IgGwas prepared as described previously (85). Alexa Fluor 488 (AF488)- andAF555–goat anti-mouse IgG, AF555–anti-mouse IgG1, and AF555–anti-mouseIgG2a, AF555-conjugated goat anti-rat IgG, AF555–goat anti-rabbit IgG, Quan-tumdot 655 (Qdot655)-streptavidin, and AF488-streptavidin were purchasedfrom Invitrogen. FITC-donkey anti-sheep IgG and FITC-donkey anti-rabbit IgGwere purchased from Jackson Immunoresearch (West Grove, PA). Anti-CD45and anti-CD90 antibody-conjugated magnetic beads were purchased from Milte-nyi Biotech (Auburn, CA).

Synthesis of high-molecular-mass sialoside-PAA probe. Biotinylated low-mo-lecular-mass (30 kDa) sialoside-polyacrylamide (PAA) conjugates bearingNeu5Ac�2-3[6-SO4]Gal�1-4[Fuc�1-3]GlcNAc (6�-sulfo-sLeX) or 9-biphenylcar-bonyl (BPC)-Neu5Ac�2-6Gal�1-4GlcNAc were synthesized by coupling spac-ered oligosaccharides and spacered biotin to poly (4-nitrophenyl acrylate) asdescribed previously (16) Biotinylated high-molecular-mass (1,000 kDa) PAA

conjugates were synthesized by coupling spacered oligosaccharides and spaceredbiotin to poly (N-oxysuccinimide acrylate) as described previously (67); the1,000-kDa lactose conjugate was obtained from The Consortium for FunctionalGlycomics.

Mice. The IL5 transgenic (IL5-Tg) mouse line NJ.1638 was the generous giftof James J. Lee (Mayo Clinic, Scottsdale, AZ) (41). C57BL/6 mice were obtainedfrom the Scripps Research Institute. Protocols were conducted in accordancewith the National Institutes of Health and the Scripps Research Institute.

Plasmids. The full-length cDNA fragment of Siglec-F and human CD22 wasamplified by PCR and cloned into pcDNA5/FRT/V5-His vector (Invitrogen)(71). Site-directed mutagenesis was performed using a Genetailor site-directedmutagenesis system (Invitrogen). The primers used for construction of Siglec-Fmutants were as follows: for T535F, 5�-GGATGAGCCTGAACTCCACTTTGCCTCCCTC-3� (sense) and 5�-GGATGAGCCTGAACTCCACTTTGCCTCCCTC-3� (antisense); for T568F, 5�-GGAGGCTATGAAATCTGTATTCACAGACATC-3� (sense) and 5�-TACAGATTTCATAGCCTCCGTGTTCTGAG-3�(antisense); and for the cytoplasmic tail deletion, dC, 5�-TTTTCACAGTGAAGGTCCTCTGATAGAAATCAGCCC-3� (sense) and 5�-GAGGACCTTCACTGTGAAAAAGATGAGGCA-3� (antisense). Dynamin-1 cDNA constructs (wildtype [WT] and K44A) were obtained from Sandra L. Schmid (The ScrippsResearch Institute, San Diego, CA). ADP ribosylation factor 6 (ARF6)-en-hanced green fluorescent protein (EGFP) cDNA constructs (WT, T27N, andQ67L) were obtained from Julie G. Donaldson (National Institutes of Health,Bethesda, MD). Cells were grown to 70 to 80% confluence on coverslips andtransiently transfected with plasmid using Lipofectamine 2000 (Invitrogen).

Cell culture. CHO cells were cultured in Ham’s F-12 medium, respectively,supplemented with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin(100 �g/ml). Stable clones of CHO cells expressing Siglec-F, Siglec-F mutants,and human CD22 were generated by Lipofectamine 2000 (Invitrogen) and se-lection in 0.5 mg/ml hygromycin B (Roche Molecular Biochemicals).

Agent treatments. Cells were preincubated for 30 min at 37°C in Ham’sF-12–10% fetal calf serum medium containing 10 mM methyl-�-cyclodextrin(CD) (Sigma-Aldrich), 5 �M latrunculin A (Sigma-Aldrich), 1 �M jasplakinolide(EMD Biosciences, CA), 100 �M genistein (Sigma-Aldrich), 100 �M PP2 (EMDBiosciences), 100 �M sodium pervanadate, 1 �M nocodazole (Sigma-Aldrich),10 �M amiloride (Sigma-Aldrich), 50 �M nystatin (Sigma-Aldrich), or 10 �Mbrefeldin A (Sigma-Aldrich). Cells were then incubated with PE–anti-Siglec-F inthe continued presence of the agents at 37°C for 1 h. Agent treatment did notaffect cell viability.

Flow cytometry. Cells (5 � 105) suspended in PBS/BSA (10 mM phosphate-buffered saline [pH 7.0] containing 10 mg/ml bovine serum albumin) were incu-bated with 10 �g/ml of primary antibody or sialoside-PAA probe for 30 min onice. After washing with PBS/BSA, cells were incubated with 10 �g/ml of FITC-secondary antibody or streptavidin-Cy5-PE for 30 min on ice. For sialidasepretreatment, cells in PBS/BSA were incubated with 25 mU of A. ureafacienssialidase (Roche Molecular Biochemicals) for 30 min at 37°C. Flow cytometrydata were acquired on a FACS Calibur flow cytometer (BD Biosciences, SanJose, CA) and analyzed using the CellQuest software.

Immunofluorescence microscopy. For the internalization assay, CHO cellscultured on coverslips for 2 days were labeled with fluorescence-conjugatedantibody (10 �g/ml) in PBS/BSA for 30 min on ice. Cells were then incubated at37°C for various times, and internalization was stopped by transferring the cellsto ice. After washing, cells were fixed with 4% paraformaldehyde (PFA; ElectronMicroscopy Services) for 15 min at room temperature. For colocalization exper-iments, cells were further permeabilized with 0.1% saponin in PBS for 20 min atroom temperature and then incubated with the desired antibody for 1 h at roomtemperature or 4°C overnight.

For eosinophil staining, eosinophils from IL5-Tg mice at 107/ml in PBS/BSAwere fixed with 2% PFA for 10 min at 4°C before staining with the desiredantibodies for 30 min at 4°C. After cell surface staining, cells were cytospun ontoglass slides and fixed with 4% PFA for 20 min at room temperature. Cells werefurther permeabilized with 0.1% saponin in PBS for 20 min at room temperatureand then incubated with anticlathrin antibody (clone x22). Cells were mountedwith Prolong gold antifade reagent (Invitrogen) and examined using a ZeissAxiovert S100TV microscope equipped with a Bio-Rad MRC 1024 confocal laserscanner. Images were collected with Bio-Rad LaserSharp 2000 software andanalyzed with Image J (v. 1.33), Zeiss LSM image, and Adobe Photoshop.

Bacteria. Cells of Neisseria meningitidis MC58 (NRCC 6200 Cap� and NRCC4728 Cap�) were grown on chocolate agar in the presence of 20 �g/ml CMP-NeuAc to increase the level of sialylation of the lipooligosaccharide (LOS). Thecells were killed by heat treatment at 68°C for 1 h. The killed cells were labeledwith FITC by resuspending them first in PBS and then mixing them with 1 mg/mlFITC in sodium bicarbonate buffer at pH 9. Cells were reacted with the dye for

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15 min and then washed extensively. Cells were observed by phase-contrastmicroscopy to ensure they were intact and to estimate the cell number.

RESULTS

Endocytosis of Siglec-F following ligation by antibody.Endocytosis of Siglec-F was assessed by monitoring the de-crease of anti-Siglec-F from the surface of eosinophils usingflow cytometry. Siglec-F was labeled with sheep anti-Si-glec-F on ice for 30 min. The temperature was raised to37°C to initiate endocytosis, and at various times, cells weretransferred to 4°C. Residual cell-surface anti-Siglec-F was thendetected by incubation with FITC–anti-sheep IgG. As shown inFig. 1a, anti-Siglec-F was internalized over time, reaching �60%internalization relative to time zero after 30 min.

CHO cells are often used to study the mechanism of endo-cytosis of immunoreceptors (14, 39, 68). Anti-Siglec-F boundto CHO cells stably expressing Siglec-F (SigF-CHO) was in-

ternalized at a rate similar to that observed for eosinophils(Fig. 1). Anti-CD22 monoclonal antibody (MAb) was similarlyinternalized from the cell surface of CD22-CHO cells, with50% internalization observed within 30 min (data not shown).

The endocytosis of Siglec-F was also assessed by monitoringthe internalization of fluorescently labeled anti-Siglec-F MAb.Eosinophils or SigF-CHO cells were stained with PE–anti-Siglec-F MAb at 4°C and warmed to 37°C for up to 60 min.Cell-associated antibody was monitored by flow cytometry be-fore (total) or after removing residual cell surface antibody bya brief low-pH wash. After incubation at 37°C for 60 min, thelevel of intracellular PE–anti-Siglec-F MAb increased gradu-ally over time (Fig. 1b and c). The combined results demon-strate that antibody ligation initiates efficient endocytosis ofSiglec-F on both mouse eosinophils and SigF-CHO cells.

Mutation of tyrosines 538 and 561 abrogates endocytosis ofSiglec-F. Siglec-F carries two potential cytoplasmic tyrosine-

FIG. 1. Siglec-F mediates endocytosis following ligation by antibody. (a) SigF-CHO cells and mouse eosinophils were incubated with sheepanti-Siglec-F pAb on ice for 30 min and allowed to internalize at 37°C for the indicated time. Cell-surface anti-Siglec-F pAb was detected byFITC–anti-sheep IgG. Data are the average standard deviation of three independent experiments. (b) SigF-CHO cells and mouse eosinophilswere incubated with PE–anti-Siglec-F (dotted line and solid line) or isotype control (shaded) for 30 min on ice. After washing, the cells wereallowed to internalize at 37°C for the indicated time. Following the incubation, cells were washed with 0.2 M glycine buffer pH 2.0 (solid line,intracellular) to remove surface-bound antibody or PBS/BSA (shaded area and dotted line, total probe) as a control. Bound or internalizedantibody was measured by flow cytometry. Representative results are shown. (c) Data are expressed as internalized mean fluorescence intensity(MFI) of PE–anti-Siglec-F measured as in panel b. Data are the average standard deviation of triplicate determinants.

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based sorting motifs (YxxL/I) at amino acids Tyr 538 and 561.To assess whether the intact motifs are required for internal-ization, we designed mutant constructs substituting phenylala-nine for tyrosine 538 and 561, either individually or in combi-nation, and a cytoplasmic deletion mutant (Fig. 2a). Theinternalization of the mutant Siglec-F constructs was com-pared to that of WT Siglec-F by flow cytometry. AlthoughPE–anti-Siglec-F MAb was efficiently internalized in CHOcells expressing WT Siglec-F, the level of internalized antibodywas significantly decreased in CHO cells expressing the dou-ble-tyrosine mutant Y538/561F (Fig. 2b). Mutation of bothtyrosines to phenylalanine reduced internalization by nearly80% (Fig. 2c). The inhibitory effect of the cytoplasmic deletion

mutant is similar to that of the double-tyrosine mutant Y538/561F (85% inhibition), suggesting that the tyrosine-based mo-tifs are dominant sorting signals. Since mutation of eitherTyr538 or Tyr561 also inhibited (66 to �69%) endocytosis ofSiglec-F, we conclude that both tyrosine-based motifs are re-quired for optimal endocytosis of Siglec-F. In contrast to themutations in the cytoplasmic domain, an Arg-to-Ala mutationof the conserved arginine residue required for sialoside bind-ing (3) showed no effect on internalization of Siglec-F, sug-gesting that ligand-binding activity is not directly involved inthe endocytosis of Siglec-F (data not shown), as reported pre-viously for endocytosis of CD22 (87).

Siglec-F is not colocalized with clathrin domains. Since theSiglec-F tyrosine-based motifs (YxxI/L) conform to the bindingsites for clathrin-associated adapter complex 2 (AP2) andCD22 had previously been demonstrated to localize predomi-nantly in clathrin domains (23), we investigated the localizationof clathrin and Siglec-F on mouse eosinophils and SigF-CHOcells. Fixed cells were first stained with anti-Siglec-F MAb andAF555-labeled (60) anti-rat IgG. Cells were then permeabil-ized and stained with anti-clathrin MAb and AF488-labeled(green) anti-mouse IgG. As shown in Fig. 3, on both mouseeosinophils and SigF-CHO cells anti-Siglec-F staining ap-peared punctate and did not overlap with anticlathrin staining.Staining of mouse eosinophils or SigF-CHO cells with anti-Siglec-F and secondary antibody prior to fixation and perme-abilization also showed no association with clathrin (data notshown).

Endocytosis of Siglec-F and CD22 results in sorting to dif-ferent intracellular compartments. To further assess the ab-sence of Siglec-F and clathrin localization, the internalizationof Siglec-F was followed by confocal fluorescence microscopyand compared to that of CD22 and transferrin (Tf) receptor,which is known to be mediated by a clathrin-mediated mech-anism (46). SigF-CHO, CD22-CHO, and parental CHO cellswere grown on coverslips and were labeled with PE–anti-Siglec-F MAb, FITC–anti-CD22 MAb, and AF488-Tf, respec-

FIG. 2. Tyrosine-based motifs are required for endocytosis ofSiglec-F. (a) Schematic representation of WT and mutant forms ofSiglec-F. The sequence at the C-terminal end is 431 SETSRGTVLGAIWGAGMALLAVCLCLIFFTVKVLRKKSALKVAATKGNHLAKNPASTINSASITSSNIALGYPIQGHLNEPGSQTQKEQPPLATVPDTQKDEPELHYASLSFQGPMPPKPQNTEAMKSVYTEIKIHKC569, where underlined letters are the transmembrane domain andtyrosines 538 and 561 are in boldface. (b) Rates of internalization ofSiglec-F for CHO cells stably expressing WT and mutant forms ofSiglec-F were compared as in Fig. 1b. Representative flow cytometrydata of the WT and the double-tyrosine mutant (Y538/561F) ofSiglec-F are shown. (c) Percent internalization is calculated as (intra-cellular mean fluorescence intensity [MFI]/total MFI) � 100 and ex-pressed as a percentage of internalization in CHO cells expressingwild-type Siglec-F. Data are the average standard deviation of trip-licate determinants.

FIG. 3. Siglec-F is not colocalized with clathrin. SigF-CHO cells ormouse eosinophils were fixed with 2% PFA before staining with mouseanti-Siglec-F MAb and AF555–anti-rat IgG. After permeabilization,cells were stained with mouse anti-clathrin MAb and AF488–anti-mouse IgG and examined by confocal microscopy. Representativeimages are shown. Bars, 10 �m.

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tively, and allowed to internalize at 37°C for various times overa 60-min period. Slides were then fixed for microscopy. Asshown in Fig. 4a, all three proteins were diffusely localized attime zero (4°C). Within 15 min of warming to 37°C, Tf wasclearly observed in early endosomes and the tubules of therecycling compartment, visible as a bright spot near the nucleusas reported previously (46). CD22 exhibited visually identicallocalization as the Tf receptor with slightly delayed kinetics. Incontrast, anti-Siglec-F endocytosed at a much reduced rate andaccumulated in intracellular compartments with a lysosomalappearance visually distinct from Tf and anti-CD22 staining.The labeling at 60 min was a result of intracellular staining forall three proteins since there was no change in appearance ifcells were subjected to a low-pH wash (0.2 M glycine, pH 2.0)prior to fixation.

To better characterize the intracellular localization of endo-cytosed CD22 and Siglec-F, we investigated their localizationin relationship to Tf, a marker for the early endosome andrecycling compartments, and anti-Syntaxin-8, a marker for lateendosomes and lysosomes (5, 55, 76). As expected, CD22 waspredominantly colocalized with Tf in early endosomes and

recycling compartments, with negligible colocalization withthe lysosomal marker Syntaxin-8 (Fig. 4b). The results areconsistent with endocytosis occurring by a clathrin-mediatedmechanism. In contrast, anti-Siglec-F was predominantly colo-calized with anti-Syntaxin-8 and another lysosomal marker,anti-LAMP2 (not shown), and exhibited minimal colocaliza-tion with Tf. Endocytosed anti-Siglec-F also partially colocal-ized with the early endosomal marker anti-EEA-1 but not withanti-clathrin or anti-AP2 (data not shown). The results indicatethat Siglec-F and CD22 are sorted to different intracellularcompartments, with Siglec-F being predominantly sorted tolysosomes.

The effect of tyrosine-based motifs on endocytosis of Siglec-Fwas confirmed by confocal fluorescence microscopy. Whileanti-Siglec-F showed predominantly intracellular punctatestaining in CHO cells expressing WT Siglec-F, cells expressingthe double-tyrosine mutant (Y538/561F) showed only diffusesurface staining that was removed with a brief low-pH wash(not shown).

A high-affinity sialoside probe competes with cis ligands andbinds to Siglec-F on native cells. Recently, Collins et al. showed

FIG. 4. Endocytosis of Siglec-F and CD22 results in sorting to different intracellular compartments. (a) CD22-CHO and SigF-CHO cells grownon coverslips were labeled with FITC–anti-CD22 and PE–anti-Siglec-F on ice for 30 min, respectively, and allowed to internalize after shifting to37°C for the indicated times. As a control, internalization of Tf is shown. (b) Cells were incubated with antibody (FITC–anti-CD22 orPE–anti-Siglec-F) and Tf (AF555-Tf or AF488-Tf) on ice for 30 min and allowed to internalize after shifting to 37°C for 30 min. Cells were alsostained with a lysosome marker, anti-Syntaxin-8. Representative images are shown. Bars, 10 �m.

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that high-affinity sialoside ligands could compete with cis ligands,bind to CD22 on native B cells, and serve as a ligand-basedmarker of CD22 endocytosis (22). The ligand was comprised of ahigh-affinity synthetic ligand of CD22, 9-biphenylcarboxyl-NeuAc�2-6Gal�1-4GlcNAc (BPC-NeuAc-LN), attached to a1,000-kDa PAA (74) backbone. To determine if CD22 andSiglec-F carry sialoside ligands to the same compartments astheir respective anti-siglec antibodies, we sought to prepare ananalogous sialoside ligand for Siglec-F. We previously reportedthat the preferred glycan ligand for Siglec-F is 6�-sulfo-sLeX

(Neu5Ac�2-3[6-SO4]Gal�1-4[Fuc�1-3]GlcNAc) (71). How-ever, Siglec-F appears to be masked to synthetic 30-kDa PAAprobes containing this sequence unless the cells are treatedwith sialidase to destroy cis ligands, a general property of thesiglec family (25, 47, 58, 59, 78). To create high-avidity probesfor Siglec-F, 6�-sulfo-sLeX was attached to the 1,000-kDa-mo-lecular-mass PAA along with 5% biotin to allow detection withstreptavidin (74). A standard enzyme-linked immunosorbentassay using immobilized Siglec-F-Fc chimera showed that theresulting 1,000-kDa 6�-sulfo-sLeX-PAA probe bound withmuch higher binding avidity than did the standard 30-kDa6�-sulfo-sLeX-PAA probe (data not shown), indicating that theincreased multivalency did increase binding avidity forSiglec-F. The 6�-sulfo-sLeX-PAA probes (30 kDa and 1,000kDa) were then compared for their abilities to compete with cisligands and bind to native eosinophils and SigF-CHO cells, asillustrated in Fig. 5a. As found earlier, the 30-kDa probe failedto bind to native cells but bound only to the sialidase-treated(asialo) cells (71). In contrast, the 1,000-kDa probe bound tonative cells as well, demonstrating that the 1,000-kDa 6�-sulfo-sLeX-PAA probe can effectively compete with cis ligands onboth mouse eosinophils and SigF-CHO cells. Blocking withanti-Siglec-F MAb inhibited binding of the sialoside probe tothe cells (Fig. 5b), indicating that the binding is not due to a

nonspecific interaction (71). No binding of a control 1,000-kDalactose-PAA probe was observed.

Siglec-F and CD22 mediate endocytosis of glycan ligands todifferent subcellular compartments. Using the high-affinitysialoside probes that can bind to Siglec-F and CD22 on nativecells, we compared their abilities to internalize and traffic theirrespective sialoside probes to intracellular compartments (Fig.6). Cells were incubated with the appropriate 1,000-kDa bio-tinylated sialoside-PAA probe on ice for 60 min followed byincubation with PE-Cy5-streptavidin on ice for an additional30 min. The cells were then shifted to 37°C, and the degree ofprobe internalization assessed before and after low-pH washwas measured by flow cytometry at various times. As shown inFig. 6a, CD22-CHO cells efficiently internalized the BPC-NeuAc-LN-PAA probe. Similarly, the 6�-sulfo-sLeX-PAAprobe was internalized by Siglec-F on SigF-CHO cells withsomewhat slower kinetics, as seen for the anti-Siglec-F anti-body (Fig. 3). No binding or internalization was observed withthe control 1,000-kDa lactose-PAA by either CD22-CHO orSiglec-F-CHO cells (data not shown). No binding or internal-ization was observed for either the 1,000-kDa BPC-NeuAc-LN-PAA or 6�-sulfo-sLeX-PAA probe with untransfectedCHO cells (data not shown).

CD22 and Siglec-F sort glycan ligands to the same intracel-lular compartments as their respective antibodies. Endocyto-sis of sialoside probes was investigated by confocal fluores-cence microscopy (Fig. 6b). Cells grown on coverslips wereincubated with the biotinylated 1,000-kDa sialoside-PAAprobe on ice for 60 min. After washing, cells were incubatedwith Qdot655-streptavidin on ice for 30 min and allowed tointernalize at 37°C. After 60 min at 37°C, CD22 had carried the1,000-kDa BPC-NeuAc-LN-PAA probe (CD22-L) to the earlyendosome/recycling compartments marked by Tf. In contrast,Siglec-F carried the 1,000-kDa 6�-sulfo-sLeX-PAA probe

FIG. 5. High-molecular-mass sialoside probe binds to Siglec-F on native cells. (a) Mouse eosinophils prepared from IL5-Tg mice or SigF-CHOcells pretreated with or without A. ureafaciens sialidase were incubated with biotinylated 1,000-kDa lactose-PAA (negative control) or 1,000-kDa6�-sulfo-sLeX-PAA probe on ice for 60 min. After washing, bound probe was detected with PE-Cy5-streptavidin and analyzed by flow cytometry.(b) Sialidase-treated mouse eosinophils were preincubated with or without anti-Siglec-F MAb and then were incubated with the biotinylatedsialoside-PAA probes on ice for 60 min. After washing, bound probe was detected with PE-Cy5-streptavidin and analyzed by flow cytometry.

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(SigF-L) to lysosomes labeled with anti-LAMP2. These resultsindicate that the glycan ligand is sorted to the same intracel-lular compartment as is antibody.

Endocytosis of Siglec-F is dynamin-1 independent. To fur-ther investigate the mechanism of endocytosis, we evaluatedthe dependence on dynamin-1. Dynamin-1 is a large GTPasethat regulates vesiculation events at the plasma membrane forseveral clathrin-dependent and clathrin-independent endocyticpathways (24). Expression of a dominant-negative dynamin-1mutant (Dyn1-K44A) inhibits both clathrin- and caveolin-me-diated endocytic pathways (28, 35, 40, 51). Internalization of Tf

and anti-CD22 was virtually inhibited by Dyn1-K44A, but notby WT dynamin-1 (Dyn1-WT), whereas that of anti-Siglec-Fwas not inhibited by the expression of either Dyn1-K44A orDyn1-WT (Fig. 7). These results indicate that endocytosis ofCD22 is dynamin dependent, characteristic of a clathrin-me-diated pathway, whereas endocytosis of Siglec-F is dynaminindependent.

FIG. 6. Siglec-F and CD22 mediate endocytosis of glycan ligands.(a) SigF-CHO cells or CD22-CHO cells were incubated with the 1,000-kDa 6�-sulfo-sLeX-PAA probe and the 1,000-kDa BPC-NeuAc-LN-PAA probe on ice for 60 min followed by incubation with PE-Cy5-streptavidin on ice for 30 min. After washing, the cells were thenshifted to 37°C for the indicated times. Following the incubation, cellswere washed with 0.2 M glycine buffer, pH 2.0, to remove surface-bound probe or PBS/BSA as a control. Bound or internalized probewas measured by flow cytometry. The 1,000-kDa lactose-PAA probewas used as a negative control. Data are expressed as internalizedmean fluorescence intensity (MFI) of the probes and the average standard deviation of triplicates. (b) Endocytosis of sialoside probeswas investigated by confocal fluorescence microscopy. CD22 carriedthe 1,000-kDa BPC-NeuAc-LN-PAA probe (CD22-L) to the earlyendosome/recycling compartments marked by Tf, whereas Siglec-Fcarried the 1,000-kDa 6�-sulfo-sLeX-PAA probe (SigF-L) to lysosomesmarked with anti-LAMP2. Representative images are shown. Bar, 10 �m.

FIG. 7. Endocytosis of Siglec-F is Dyn1 independent. (a) SigF-CHO cells were transiently transfected with Dyn1-WT or Dyn1-K44A,both tagged with hemagglutinin (HA). Cells were incubated with Tf orPE–anti-Siglec-F for 60 min, fixed, and stained with anti-HA to detecttransfected cells. Representative images are shown. Bar, 10 �m. (b)SigF-CHO and CD22-CHO cells were transfected with Dyn1-WT orDyn1-K44A and analyzed for internalization of Siglec-F and Tf recep-tor as for panel a and for CD22 with FITC–anti-CD22. Data areexpressed as the percentage of transfected cells with Dyn1-K44A rel-ative to Dyn1-WT that contained internalized Tf, anti-CD22, or anti-Siglec-F. More than 50 transfected cells were counted. Data are theaverage standard deviation of three independent transfections. Ex-pression of Dyn1-K44A inhibited endocytosis of Tf and anti-CD22 butnot endocytosis of Siglec-F.

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Endocytosis of Siglec-F is ARF6 dependent. ARF6 is a smallGTPase that regulates the movement of endosomes betweenthe plasma membrane and a non-clathrin-derived endosomalcompartment (56). ARF6 regulates clathrin- and caveolin-in-dependent endocytosis (2, 4, 17, 48, 49, 56). Trafficking of thesemolecules is blocked by a constitutively active ARF6 mutant(ARF6-Q67L) and results in accumulation in vacuole-likestructures (17). Although expression of WT and a constitu-tively inactive ARF6 mutant (ARF6-T27N) had no effect onthe internalization of anti-Siglec-F, expression of ARF6-Q67Linhibited trafficking of anti-Siglec-F to the lysosomal compart-ment (Fig. 8). Instead, anti-Siglec-F appears to be associatedwith vacuole-like structure, indicating that endocytosis ofSiglec-F is affected by ARF6. Recently, flotillin-1 was reportedto define a clathrin-independent endocytic pathway (33). How-ever, no colocalization or internalized anti-Siglec-F with anti-flotillin-1 was observed (data not shown).

Cholesterol, tyrosine kinases, and intact actin cytoskeltonare required for endocytosis of Siglec-F. To better understandthe mechanism of endocytosis of Siglec-F, we investigated theeffects of various agents known to affect endocytic processes(Fig. 9). SigF-CHO cells were pretreated with the agent at37°C for 30 min. PE–anti-Siglec-F was then allowed to inter-nalize at 37°C for 1 h in the continued presence of the agent.After residual cell surface antibody was removed by a brieflow-pH wash (0.2 M glycine buffer, pH 2.0), the level of inter-nalized PE–anti-Siglec-F was compared to the level in un-treated cells, as shown in Fig. 9a for the cholesterol depletionagent CD. For each agent, the degree of internalization rela-tive to no agent (using mean channel fluorescence) is summa-rized in Fig. 9b and c. The CD treatment showed 92% inhibi-tion of internalization relative to the internalization in

untreated cells (Fig. 9b), suggesting that cholesterol is requiredfor internalization of Siglec-F. However, the cholesterol-se-questering agent nystatin, which inhibits endocytic pathwaysinvolving lipid rafts and caveolin, caused no inhibition at aconcentration of 50 �g/ml (61). Inhibitors of actin cytoskeletonpolymerization (latrunculin A and jasplakinolide) and micro-tuble polymerization (nocodazole) showed partial (40 to 50%)inhibition. Although genistein (general tyrosine kinase inhibi-tor) strongly inhibited internalization (87% inhibition), little orno effect was observed by PP2 (Src family kinase inhibitor).Amiloride, a macropinocytosis inhibitor, showed no inhibitoryeffect, rather increased the internalization of Siglec-F. In CHOcells, clathrin-mediated endocytosis is known to be insensitiveto treatments with CD and genistein, so inhibition of Siglec-Fendocytosis by these agents is further evidence that the processis clathrin independent (21). Similarly, caveolin-mediated en-docytosis is sensitive to the treatment with PP2 (21), so lack ofinhibition of Siglec-F endocytosis by PP2 supports a caveolin-independent mechanism.

Endocytic pathway of Siglec-F can mediate uptake ofsialylated bacteria. Having shown that Siglec-F mediates inter-nalization of antibody and synthetic sialoside probe, we inves-tigated whether the endocytic machinery of Siglec-F expressedin nonphagoctic CHO cells can mediate uptake of Neisseriameningitidis MC58 with (MC58cap�) or without (MC58cap�)capsule. MC58cap� expresses LOS (31) with a terminalNeuAc�2-3Gal�1-4GlcNAc sequence, whereas MC58cap� ex-presses, in addition to the LOS, a polysialic acid capsule[(NeuAc�2-8NeuAc)n] (80). The former sequence is recog-nized as a weak ligand by Siglec-F, while the latter is notrecognized (3, 71). SigF-CHO cells pretreated with or withoutsialidase were incubated with fixed FITC-labeled bacteria onice for 30 min and allowed to internalize at 37°C for 60 min.Noninternalized bacteria were removed with PBS/BSA washand were confirmed to be internalized by resistance to ethidiumbromide quenching of fluorescence (not shown). While neitherthe MC58cap� or MC58cap� bacteria were internalized in un-treated SigF-CHO cells (Fig. 10), MC58cap� was selectively in-ternalized by cells treated with sialidase treatment to destroy cisinteractions of Siglec-F with the NeuAc�2-3Gal linkages knownto be expressed by CHO cells. No internalization of N. meningi-tidis was observed in control CHO cells. These results indicatethat endocytic machinery of Siglec-F is capable of internalizingbacteria containing a terminal sialoside sequence on a cell surfaceglycolipid that is recognized as a ligand.

DISCUSSION

While the siglec proteins are best known as a family of sialicacid-specific lectins that regulate cell-signaling receptors andmediate cell-cell interactions (25, 26, 78), they are increasinglyrecognized for endocytic activity that can link to their cellsurface functions (9, 22, 50, 57, 64, 75, 77, 81, 84, 87). Resultspresented in this report confirm and extend the suggestion thatendocytosis of CD22 occurs by a clathrin-dependent mecha-nism mediated by tyrosine-based motifs (22, 34, 37) and showthat CD22 is transported to early endosomes and para-Golgirecycling compartments. In contrast, the CD33-related siglec,Siglec-F, undergoes endocytosis dependent on intact cytoplas-mic tyrosine residues, but by a clathrin-independent mecha-

FIG. 8. Endocytosis of Siglec-F is ARF6 dependent. SigF-CHOcells were transfected by cDNAs coding for ARF6-WT, ARF6-T27N,or ARF6-Q67L, all tagged with EGFP. Cells were allowed to internal-ize PE–anti-Siglec-F at 37°C for 60 min. Expression of ARF6-Q67Lresulted in accumulation of anti-Siglec-F in vacuole-like structures.Representative images are shown. Bars, 10 �m.

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nism, and is sorted to lysosomes. The different endocytic mech-anisms of these two siglec proteins reflect the divergence intheir functions in B cells and eosinophils, respectively.

Anti-CD22 antibody and sialoside ligands endocytosed byCD22 colocalize to early endosomes and recycling compart-ments with the Tf receptor, well documented to be an inter-nalized clathrin-dependent mechanism (46). Moreover, endo-cytosis is dependent on the dynamin-1 GTPase required forclathrin-mediated vesicle formation (24). Clathrin-dependent

endocytosis of CD22 is consistent with its activity as a regulatorof BCR signaling since the endocytosis of the BCR is alsopredominantly clathrin dependent and required for dampeningof BCR signaling (70). It is of interest that the CD22 ITIMsinvolved in recruitment of SHP-1 are the same tyrosine motifsthat mediate binding of the clathrin adaptor protein AP2 (37).However, while tyrosine phosphorylation is required for SHP-1binding, phosphorylation blocks binding of AP2, suggesting aninverse regulation of these two activities (37).

FIG. 9. Endocytosis of Siglec-F requires cholesterol, tyrosine kinases, and actin cytoskeleton. SigF-CHO cells were left untreated (control) orpretreated with the indicated agents at 37°C for 30 min, and internalization of PE–anti-Siglec-F was measured by flow cytometry as in Fig. 1b andc. Representative flow cytometry data are shown in panel a. (b) Percent internalization is calculated as (intracellular mean fluorescence intensity[MFI]/total MFI) � 100 and expressed as a percentage of internalization in control cells. Data are the average standard deviation of triplicatedeterminants. (c) Summary of the effects of the agent on endocytosis of Siglec-F.

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Rapid mobilization of CD22 to the cell surface followingBCR ligation is consistent with redistribution of an intracellu-lar pool of CD22 localized in the para-Golgi recycling com-partment shared by other recycling receptors (82). Conversely,CD22 antibody induces rapid endocytosis of CD22 to the samecompartment, reaching a plateau of 60 to 70% internalized.This redistribution is achieved without increased degradation(64, 87) and has been suggested to be consistent with redistri-bution of CD22 to an intracellular recycling pool (73). Elegantstudies by Shan and Press (64) earlier suggested that the con-stitutive endocytosis of CD22 was terminal, leading to degra-dation with a half-life of �8 h without recycling to the cellsurface. However, given the slower kinetics of CD22 endocy-tosis relative to the Tf receptor (Fig. 4a), recycling of CD22from the intracellular pool might also be slower, and it ispossible that recycling was not observed over the time coursesufficient to demonstrate recycling of the Tf receptor.

In contrast to CD22, Siglec-F endocytosis traffics to lysoso-mal compartments by a mechanism that is clathrin, caveolin,and dynamin-1 independent. The endocytic pathway utilizedby Siglec-F appears to be similar to that utilized by majorhistocompatibility complex class 1 (MHC-1), and several otherleukocyte receptors including interleukin-2 receptor � subunit(Tac), E-cadherin, integrins, and CD59 (2, 4, 17, 48, 49, 56). Inthis pathway, receptors are first sorted to ARF6-positive en-dosomes, which then fuse with early endosomes derived fromclathrin-cargo, before being sorted to late endosomes and ly-sosomes. As shown for Siglec-F (Fig. 6), expression of theARF6-Q67L mutant, which blocks fusion of ARF6-positiveendosomes with early endosomes, results in the accumulationof receptors in vacuole-like structures (49). The requirementfor tyrosine-dependent endocytosis of Siglec-F has also beendemonstrated for MHC-1 (42, 63), although the exact role ofthis sequence in the endocytic mechanism remains to be de-fined.

As recently shown by Zhang et al. (86), Siglec-F negativelyregulates eosinophil responses to allergens. The endocytic

pathway utilized by Siglec-F is also suggestive of receptorswhose function is to deliver cargo to lysosomes for degradationand thus may also function as an innate immune receptorrelated to bacterial clearance or antigen presentation, as pro-posed for Siglec-H, another CD33-related siglec expressed onplasmacytoid dendritic cell precursors (84). Clinical and exper-imental investigations have shown that eosinophils can func-tion as antigen-presenting cells that can process microbial,viral, and parasitic antigens and migrate to lymph nodes topresent antigens to CD4� T cells, thereby promoting T-cellproliferation and polarization (29, 44, 65, 66). It was reportedrecently that Siglec-F is highly expressed on CD11c� lungmacrophages (52). We also found that Siglec-F is expressed onalveolar macrophages, bone marrow-derived dendritic cells,and bone marrow-derived macrophages (data not shown).

Recent reports on other members of the siglec family alsosuggest that they may function as receptors for clearance ofpathogens as part of the innate immune response. For exam-ple, Sn and Siglec-5 bind to and internalize the invasive humanpathogen Neisseria meningitidis, expressing sialylated lipopoly-saccharides containing the NeuAc�2-3Gal linkage in a sialicacid-dependent manner even in the presence of cis ligands(38). As shown in this report, Siglec-F can also mediate uptakeof Neisseria meningitidis expressing sialylated lipooligosaccha-rides containing the NeuAc�2-3Gal linkage recognized as aligand by Siglec-F (3, 71). Sn was also found to mediate asso-ciation of macrophages with Trypanosoma cruzi, a parasiteexpressing cell surface sialic acids (45), and porcine Sn wasshown to mediate the entry of porcine reproductive and respi-ratory viruses into porcine alveolar macrophages (30, 77).Thus, bacteria, parasites, and viruses expressing sialic acids ontheir cell surfaces are likely to be natural trans ligands of siglecproteins (20, 79). The diversity of interactions of siglec proteinswith sialylated pathogens suggests that they may play multipleroles in their interactions with immune cells (8, 18, 30, 38, 45).

The roles emerging for the rapidly evolving siglec familyreflect diverse functions resulting from their varied specificityfor sialylated ligands by the N-terminal Ig domain and regula-tory motifs in their cytoplasmic domains that regulate cellsignaling receptors and endocytosis. As documented in thisreport, two siglec proteins of unrelated function, CD22 andSiglec-F, exhibit clathrin-dependent and clathrin-independentmechanisms of endocytosis, both of which require intact cyto-plasmic tyrosine-based motifs.

Since Siglec-F is a typical CD33-related siglec that shareshighly conserved cytoplasmic tyrosine-based motifs, we predictthat other CD33-related siglec proteins show similar endocyticproperties to Siglec-F. While there is no clear human orthologof Siglec-F, human Siglec-8 is considered a functionally con-vergent paralog due to its restricted expression on eosinophilsand unique specificity for the sialoside ligand 6�-sulfo-sLeX

(11, 71). Thus, it will be of particular interest to see if thefunctional convergence of Siglec-F and Siglec-8 extends totheir mechanisms of endocytosis. However, it is premature togeneralize the mechanisms of endocytosis for the siglec family.For example, some CD33-related siglec proteins such asSiglec-H and Siglec-14 lack cytoplasmic tyrosine-based motifs,but they associate with the DAP12 coreceptor that contains acytoplasmic immunoreceptor tyrosine-based activation motif(ITAM) (10, 84). Thus, siglec proteins may undergo endocy-

FIG. 10. The endocytic machinery of Siglec-F can mediate uptakeof sialylated bacteria. SigF-CHO cells grown on coverslips were pre-treated with or without Arthrobacter ureafaciens sialidase and wereincubated with fixed, FITC-labeled Neisseria meningitidis MC58cap�

containing the NeuAc�2-3Gal sequence on ice for 30 min and allowedto internalize at 37°C for 60 min. Noninternalized bacteria were re-moved with a PBS/BSA wash. Cells were examined by confocal mi-croscopy. Representative images are shown. Bar, 10 �m.

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tosis by yet other mechanisms than those of CD22 and Siglec-Fdescribed here. It is likely that a better understanding of theendocytic mechanisms of the siglec proteins will provide in-sights into their biological roles.

ACKNOWLEDGMENTS

We thank Jill Ferguson, Mary O’Reilly, and Anna Tran-Crie forhelp in preparing this article. We are grateful to Sandra L. Schmid fordynamin-1 cDNA constructs (WT and the K44A mutant), discussions,and suggestions; Julie G. Donaldson for ARF6-EGFP cDNA con-structs (WT and the T27N and Q67L mutants); James J. Lee forIL5-Tg mice; and The Consortium for Functional Glycomics for the1,000-kDa lactose-PAA probe.

This work was supported by grants GM60938 and AI050143 fromthe National Institutes of Health, by a Wellcome Trust Senior Fellow-ship awarded to P.R.C., by Presidium RAS “Molecular and Cell Biol-ogy” to N.V.B., and by a JSPS Postdoctoral Fellowship for ResearchAbroad awarded to H.T.

REFERENCES

1. Ahluwalia, J., A. Tinker, L. H. Clapp, M. R. Duchen, A. Y. Abramov, S. Pope,M. Nobles, and A. W. Segal. 2004. The large-conductance Ca2�-activatedK� channel is essential for innate immunity. Nature 427:853–858.

2. Aikawa, Y., and T. F. Martin. 2003. ARF6 regulates a plasma membranepool of phosphatidylinositol(4,5)bisphosphate required for regulated exocy-tosis. J. Cell Biol. 162:647–659.

3. Angata, T., R. Hingorani, N. M. Varki, and A. Varki. 2001. Cloning andcharacterization of a novel mouse Siglec, mSiglec-F: differential evolution ofthe mouse and human (CD33) Siglec-3-related gene clusters. J. Biol. Chem.276:45128–45136.

4. Arnaoutova, I., C. L. Jackson, O. S. Al-Awar, J. G. Donaldson, and Y. P. Loh.2003. Recycling of Raft-associated prohormone sorting receptor car-boxypeptidase E requires interaction with ARF6. Mol. Biol. Cell 14:4448–4457.

5. Augustin, R., J. Riley, and K. H. Moley. 2005. GLUT8 contains a [DE]XXX-L[LI] sorting motif and localizes to a late endosomal/lysosomal compart-ment. Traffic 6:1196–1212.

6. Avril, T., H. Floyd, F. Lopez, E. Vivier, and P. R. Crocker. 2004. Themembrane-proximal immunoreceptor tyrosine-based inhibitory motif is crit-ical for the inhibitory signaling mediated by Siglecs-7 and -9, CD33-relatedSiglecs expressed on human monocytes and NK cells. J. Immunol. 173:6841–6849.

7. Avril, T., S. D. Freeman, H. Attrill, R. G. Clarke, and P. R. Crocker. 2005.Siglec-5 (CD170) can mediate inhibitory signaling in the absence of immu-noreceptor tyrosine-based inhibitory motif phosphorylation. J. Biol. Chem.280:19843–19851.

8. Avril, T., E. R. Wagner, H. J. Willison, and P. R. Crocker. 2006. Sialicacid-binding immunoglobulin-like lectin 7 mediates selective recognition ofsialylated glycans expressed on Campylobacter jejuni lipooligosaccharides.Infect. Immun. 74:4133–4141.

9. Biedermann, B., D. Gil, D. T. Bowen, and P. R. Crocker. 2007. Analysis of theCD33-related siglec family reveals that Siglec-9 is an endocytic receptorexpressed on subsets of acute myeloid leukemia cells and absent from nor-mal hematopoietic progenitors. Leuk. Res. 31:211–220.

10. Blasius, A. L., M. Cella, J. Maldonado, T. Takai, and M. Colonna. 2006.Siglec-H is an IPC-specific receptor that modulates type I IFN secretionthrough DAP12. Blood 107:2474–2476.

11. Bochner, B. S., R. A. Alvarez, P. Mehta, N. V. Bovin, O. Blixt, J. R. White,and R. L. Schnaar. 2005. Glycan array screening reveals a candidate ligandfor Siglec-8. J. Biol. Chem. 280:4307–4312.

12. Bonazzi, M., and P. Cossart. 2006. Bacterial entry into cells: a role for theendocytic machinery. FEBS Lett. 580:2962–2967.

13. Bonifacino, J. S., and L. M. Traub. 2003. Signals for sorting of transmem-brane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72:395–447.

14. Booth, J. W., M. K. Kim, A. Jankowski, A. D. Schreiber, and S. Grinstein.2002. Contrasting requirements for ubiquitylation during Fc receptor-medi-ated endocytosis and phagocytosis. EMBO J. 21:251–258.

15. Borelli, V., F. Vita, S. Shankar, M. R. Soranzo, E. Banfi, G. Scialino, C.Brochetta, and G. Zabucchi. 2003. Human eosinophil peroxidase inducessurface alteration, killing, and lysis of Mycobacterium tuberculosis. Infect.Immun. 71:605–613.

16. Bovin, N. V., E. Korchagina, T. V. Zemlyanukhina, N. E. Byramova, O. E.Galanina, A. E. Zemlyakov, A. E. Ivanov, V. P. Zubov, and L. V. Mochalova.1993. Synthesis of polymeric neoglycoconjugates based on N-substitutedpolyacrylamides. Glycoconj. J. 10:142–151.

17. Brown, F. D., A. L. Rozelle, H. L. Yin, T. Balla, and J. G. Donaldson. 2001.

Phosphatidylinositol 4,5-bisphosphate and Arf6-regulated membrane traffic.J. Cell Biol. 154:1007–1017.

18. Carlin, A. F., A. L. Lewis, A. Varki, and V. Nizet. 2007. Group B strepto-coccal capsular sialic acids interact with Siglecs (immunoglobulin-like lec-tins) on human leukocytes. J. Bacteriol. 189:1231–1237.

19. Castro, A. G., N. Esaguy, P. M. Macedo, A. P. Aguas, and M. T. Silva. 1991.Live but not heat-killed mycobacteria cause rapid chemotaxis of large num-bers of eosinophils in vivo and are ingested by the attracted granulocytes.Infect. Immun. 59:3009–3014.

20. Chava, A. K., S. Bandyopadhyay, M. Chatterjee, and C. Mandal. 2004.Sialoglycans in protozoal diseases: their detection, modes of acquisition andemerging biological roles. Glycoconj. J. 20:199–206.

21. Cheng, Z. J., R. D. Singh, D. K. Sharma, E. L. Holicky, K. Hanada, D. L.Marks, and R. E. Pagano. 2006. Distinct mechanisms of clathrin-indepen-dent endocytosis have unique sphingolipid requirements. Mol. Biol. Cell17:3197–3210.

22. Collins, B. E., O. Blixt, S. Han, B. Duong, H. Li, J. K. Nathan, N. Bovin, andJ. C. Paulson. 2006. High-affinity ligand probes of CD22 overcome thethreshold set by cis ligands to allow for binding, endocytosis, and killing of Bcells. J. Immunol. 177:2994–3003.

23. Collins, B. E., B. A. Smith, P. Bengtson, and J. C. Paulson. 2006. Ablationof CD22 in ligand-deficient mice restores B cell receptor signaling. Nat.Immunol. 7:199–206.

24. Conner, S. D., and S. L. Schmid. 2003. Regulated portals of entry into thecell. Nature 422:37–44.

25. Crocker, P. R. 2005. Siglecs in innate immunity. Curr. Opin. Pharmacol.5:431–437.

26. Crocker, P. R. 2002. Siglecs: sialic-acid-binding immunoglobulin-like lectinsin cell-cell interactions and signalling. Curr. Opin. Struct. Biol. 12:609–615.

27. Crocker, P. R., J. C. Paulson, and A. Varki. 2007. Siglecs and their roles inthe immune system. Nat. Rev. Immunol. 7:255–266.

28. Damke, H., D. D. Binns, H. Ueda, S. L. Schmid, and T. Baba. 2001. DynaminGTPase domain mutants block endocytic vesicle formation at morphologi-cally distinct stages. Mol. Biol. Cell 12:2578–2589.

29. Del Pozo, V., B. De Andres, E. Martin, B. Cardaba, J. C. Fernandez, S.Gallardo, P. Tramon, F. Leyva-Cobian, P. Palomino, and C. Lahoz. 1992.Eosinophil as antigen-presenting cell: activation of T cell clones and T cellhybridoma by eosinophils after antigen processing. Eur. J. Immunol. 22:1919–1925.

30. Delputte, P. L., and H. J. Nauwynck. 2004. Porcine arterivirus infection ofalveolar macrophages is mediated by sialic acid on the virus. J. Virol. 78:8094–8101.

31. Dobrina, A., R. Menegazzi, T. M. Carlos, E. Nardon, R. Cramer, T. Zacchi,J. M. Harlan, and P. Patriarca. 1991. Mechanisms of eosinophil adherenceto cultured vascular endothelial cells. Eosinophils bind to the cytokine-induced ligand vascular cell adhesion molecule-1 via the very late activationantigen-4 integrin receptor. J. Clin. Investig. 88:20–26.

32. Galioto, A. M., J. A. Hess, T. J. Nolan, G. A. Schad, J. J. Lee, and D.Abraham. 2006. Role of eosinophils and neutrophils in innate and adaptiveprotective immunity to larval Strongyloides stercoralis in mice. Infect. Immun.74:5730–5738.

33. Glebov, O. O., N. A. Bright, and B. J. Nichols. 2006. Flotillin-1 defines aclathrin-independent endocytic pathway in mammalian cells. Nat. Cell Biol.8:46–54.

34. Grewal, P. K., M. Boton, K. Ramirez, B. E. Collins, A. Saito, R. S. Green, K.Ohtsubo, D. Chui, and J. D. Marth. 2006. ST6Gal-I restrains CD22-depen-dent antigen receptor endocytosis and Shp-1 recruitment in normal andpathogenic immune signaling. Mol. Cell. Biol. 26:4970–4981.

35. Henley, J. R., E. W. Krueger, B. J. Oswald, and M. A. McNiven. 1998.Dynamin-mediated internalization of caveolae. J. Cell Biol. 141:85–99.

36. Inoue, Y., Y. Matsuwaki, S. H. Shin, J. U. Ponikau, and H. Kita. 2005.Nonpathogenic, environmental fungi induce activation and degranulation ofhuman eosinophils. J. Immunol. 175:5439–5447.

37. John, B., B. R. Herrin, C. Raman, Y. N. Wang, K. R. Bobbitt, B. A. Brody,and L. B. Justement. 2003. The B cell coreceptor CD22 associates withAP50, a clathrin-coated pit adapter protein, via tyrosine-dependent interac-tion. J. Immunol. 170:3534–3543.

38. Jones, C., M. Virji, and P. R. Crocker. 2003. Recognition of sialylatedmeningococcal lipopolysaccharide by siglecs expressed on myeloid cells leadsto enhanced bacterial uptake. Mol. Microbiol. 49:1213–1225.

39. Keller, G. A., M. W. Siegel, and I. W. Caras. 1992. Endocytosis of glyco-phospholipid-anchored and transmembrane forms of CD4 by different en-docytic pathways. EMBO J. 11:863–874.

40. Le, P. U., G. Guay, Y. Altschuler, and I. R. Nabi. 2002. Caveolin-1 is anegative regulator of caveolae-mediated endocytosis to the endoplasmicreticulum. J. Biol. Chem. 277:3371–3379.

41. Lee, N. A., M. P. McGarry, K. A. Larson, M. A. Horton, A. B. Kristensen,and J. J. Lee. 1997. Expression of IL-5 in thymocytes/T cells leads to thedevelopment of a massive eosinophilia, extramedullary eosinophilopoiesis,and unique histopathologies. J. Immunol. 158:1332–1344.

42. Lizee, G., G. Basha, J. Tiong, J. P. Julien, M. Tian, K. E. Biron, and W. A.Jefferies. 2003. Control of dendritic cell cross-presentation by the major

VOL. 27, 2007 ENDOCYTIC MECHANISMS OF SIGLEC PROTEINS 5709

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/jour

nal/m

cb o

n 04

Feb

ruar

y 20

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8.14

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3.

histocompatibility complex class I cytoplasmic domain. Nat. Immunol.4:1065–1073.

43. Lock, K., J. Zhang, J. Lu, S. H. Lee, and P. R. Crocker. 2004. Expression ofCD33-related siglecs on human mononuclear phagocytes, monocyte-deriveddendritic cells and plasmacytoid dendritic cells. Immunobiology 209:199–207.

44. Mawhorter, S. D., J. W. Kazura, and W. H. Boom. 1994. Human eosinophilsas antigen-presenting cells: relative efficiency for superantigen- and antigen-induced CD4� T-cell proliferation. Immunology 81:584–591.

45. Monteiro, V. G., C. S. Lobato, A. R. Silva, D. V. Medina, M. A. de Oliveira,S. H. Seabra, W. de Souza, and R. A. DaMatta. 2005. Increased associationof Trypanosoma cruzi with sialoadhesin positive mice macrophages. Parasi-tol. Res. 97:380–385.

46. Mukherjee, S., R. N. Ghosh, and F. R. Maxfield. 1997. Endocytosis. Physiol.Rev. 77:759–803.

47. Nakamura, K., T. Yamaji, P. R. Crocker, A. Suzuki, and Y. Hashimoto. 2002.Lymph node macrophages, but not spleen macrophages, express high levelsof unmasked sialoadhesin: implication for the adhesive properties of mac-rophages in vivo. Glycobiology 12:209–216.

48. Naslavsky, N., R. Weigert, and J. G. Donaldson. 2004. Characterization of anonclathrin endocytic pathway: membrane cargo and lipid requirements.Mol. Biol. Cell 15:3542–3552.

49. Naslavsky, N., R. Weigert, and J. G. Donaldson. 2003. Convergence ofnon-clathrin- and clathrin-derived endosomes involves Arf6 inactivation andchanges in phosphoinositides. Mol. Biol. Cell 14:417–431.

50. Nguyen, D. H., E. D. Ball, and A. Varki. 2006. Myeloid precursors and acutemyeloid leukemia cells express multiple CD33-related Siglecs. Exp. Hema-tol. 34:728–735.

51. Oh, P., D. P. McIntosh, and J. E. Schnitzer. 1998. Dynamin at the neck ofcaveolae mediates their budding to form transport vesicles by GTP-drivenfission from the plasma membrane of endothelium. J. Cell Biol. 141:101–114.

52. Padigel, U. M., J. J. Lee, T. J. Nolan, G. A. Schad, and D. Abraham. 2006.Eosinophils can function as antigen-presenting cells to induce primary andsecondary immune responses to Strongyloides stercoralis. Infect. Immun. 74:3232–3238.

53. Paul, S. P., L. S. Taylor, E. K. Stansbury, and D. W. McVicar. 2000. Myeloidspecific human CD33 is an inhibitory receptor with differential ITIM func-tion in recruiting the phosphatases SHP-1 and SHP-2. Blood 96:483–490.

54. Pierce, S. K. 2002. Lipid rafts and B-cell activation. Nat. Rev. Immunol.2:96–105.

55. Prekeris, R., B. Yang, V. Oorschot, J. Klumperman, and R. H. Scheller.1999. Differential roles of syntaxin 7 and syntaxin 8 in endosomal trafficking.Mol. Biol. Cell 10:3891–3908.

56. Radhakrishna, H., and J. G. Donaldson. 1997. ADP-ribosylation factor 6 reg-ulates a novel plasma membrane recycling pathway. J. Cell Biol. 139:49–61.

57. Rapoport, E. M., Y. B. Sapot’ko, G. V. Pazynina, V. K. Bojenko, and N. V.Bovin. 2005. Sialoside-binding macrophage lectins in phagocytosis of apop-totic bodies. Biochemistry (Moscow) 70:330–338.

58. Razi, N., and A. Varki. 1999. Cryptic sialic acid binding lectins on humanblood leukocytes can be unmasked by sialidase treatment or cellular activa-tion. Glycobiology 9:1225–1234.

59. Razi, N., and A. Varki. 1998. Masking and unmasking of the sialic acid-binding lectin activity of CD22 (Siglec-2) on B lymphocytes. Proc. Natl.Acad. Sci. USA 95:7469–7474.

60. Rodal, S. K., G. Skretting, O. Garred, F. Vilhardt, B. van Deurs, and K.Sandvig. 1999. Extraction of cholesterol with methyl-beta-cyclodextrin per-turbs formation of clathrin-coated endocytic vesicles. Mol. Biol. Cell 10:961–974.

61. Rothberg, K. G., J. E. Heuser, W. C. Donzell, Y. S. Ying, J. R. Glenney, andR. G. Anderson. 1992. Caveolin, a protein component of caveolae membranecoats. Cell 68:673–682.

62. Rothenberg, M. E., and S. P. Hogan. 2006. The eosinophil. Annu. Rev.Immunol. 24:147–174.

63. Santos, S. G., A. N. Antoniou, P. Sampaio, S. J. Powis, and F. A. Arosa. 2006.Lack of tyrosine 320 impairs spontaneous endocytosis and enhances releaseof HLA-B27 molecules. J. Immunol. 176:2942–2949.

64. Shan, D., and O. W. Press. 1995. Constitutive endocytosis and degradationof CD22 by human B cells. J. Immunol. 154:4466–4475.

65. Shi, H. Z. 2004. Eosinophils function as antigen-presenting cells. J. Leukoc.Biol. 76:520–527.

66. Shi, H. Z., A. Humbles, C. Gerard, Z. Jin, and P. F. Weller. 2000. Lymphnode trafficking and antigen presentation by endobronchial eosinophils.J. Clin. Investig. 105:945–953.

67. Shilova, N. V., O. E. Galanina, T. V. Pochechueva, A. A. Chinarev, V. A.Kadykov, A. B. Tuzikov, and N. V. Bovin. 2005. High molecular weightneoglycoconjugates for solid phase assays. Glycoconj. J. 22:43–51.

68. Sigismund, S., T. Woelk, C. Puri, E. Maspero, C. Tacchetti, P. Transidico,P. P. Di Fiore, and S. Polo. 2005. Clathrin-independent endocytosis ofubiquitinated cargos. Proc. Natl. Acad. Sci. USA 102:2760–2765.

69. Stoddart, A., M. L. Dykstra, B. K. Brown, W. Song, S. K. Pierce, and F. M.Brodsky. 2002. Lipid rafts unite signaling cascades with clathrin to regulateBCR internalization. Immunity 17:451–462.

70. Stoddart, A., A. P. Jackson, and F. M. Brodsky. 2005. Plasticity of B cellreceptor internalization upon conditional depletion of clathrin. Mol. Biol.Cell 16:2339–2348.

71. Tateno, H., P. R. Crocker, and J. C. Paulson. 2005. Mouse Siglec-F andhuman Siglec-8 are functionally convergent paralogs that are selectivelyexpressed on eosinophils and recognize 6�-sulfo-sialyl Lewis X as a preferredglycan ligand. Glycobiology 15:1125–1135.

72. Taylor, V. C., C. D. Buckley, M. Douglas, A. J. Cody, D. L. Simmons, andS. D. Freeman. 1999. The myeloid-specific sialic acid-binding receptor,CD33, associates with the protein-tyrosine phosphatases, SHP-1 and SHP-2.J. Biol. Chem. 274:11505–11512.

73. Tedder, T. F., J. C. Poe, and K. M. Haas. 2005. CD22: a multifunctionalreceptor that regulates B lymphocyte survival and signal transduction. Adv.Immunol. 88:1–50.

74. Toppila, S., T. Paavonen, A. Laitinen, L. A. Laitinen, and R. Renkonen. 2000.Endothelial sulfated sialyl Lewis x glycans, putative L-selectin ligands, arepreferentially expressed in bronchial asthma but not in other chronic inflam-matory lung diseases. Am. J. Respir. Cell Mol. Biol. 23:492–498.

75. Tuscano, J. M., R. T. O’Donnell, L. A. Miers, L. A. Kroger, D. L. Kukis, K. R.Lamborn, T. F. Tedder, and G. L. DeNardo. 2003. Anti-CD22 ligand-block-ing antibody HB22.7 has independent lymphomacidal properties and aug-ments the efficacy of 90Y-DOTA-peptide-Lym-1 in lymphoma xenografts.Blood 101:3641–3647.

76. Uthayakumar, S., and B. L. Granger. 1995. Cell surface accumulation ofoverexpressed hamster lysosomal membrane glycoproteins. Cell Mol. Biol.Res. 41:405–420.

77. Vanderheijden, N., P. L. Delputte, H. W. Favoreel, J. Vandekerckhove, J.Van Damme, P. A. van Woensel, and H. J. Nauwynck. 2003. Involvement ofsialoadhesin in entry of porcine reproductive and respiratory syndrome virusinto porcine alveolar macrophages. J. Virol. 77:8207–8215.

78. Varki, A., and T. Angata. 2006. Siglecs—the major subfamily of I-type lec-tins. Glycobiology 16:1R–27R.

79. Vimr, E. R., K. A. Kalivoda, E. L. Deszo, and S. M. Steenbergen. 2004.Diversity of microbial sialic acid metabolism. Microbiol. Mol. Biol. Rev.68:132–153.

80. Wakarchuk, W., A. Martin, M. P. Jennings, E. R. Moxon, and J. C. Richards.1996. Functional relationships of the genetic locus encoding the glycosyl-transferase enzymes involved in expression of the lacto-N-neotetraose ter-minal lipopolysaccharide structure in Neisseria meningitidis. J. Biol. Chem.271:19166–19173.

81. Walter, R. B., B. W. Raden, D. M. Kamikura, J. A. Cooper, and I. D.Bernstein. 2005. Influence of CD33 expression levels and ITIM-dependentinternalization on gemtuzumab ozogamicin-induced cytotoxicity. Blood 105:1295–1302.

82. Yamashiro, D. J., B. Tycko, S. R. Fluss, and F. R. Maxfield. 1984. Segrega-tion of transferrin to a mildly acidic (pH 6.5) para-Golgi compartment in therecycling pathway. Cell 37:789–800.

83. Yazdanbakhsh, M., C. M. Eckmann, A. A. M. Bot, and D. Roos. 1986.Bactericidal action of eosinophils from normal human blood. Infect. Immun.53:192–198.

84. Zhang, J., A. Raper, N. Sugita, R. Hingorani, M. Salio, M. J. Palmowski, V.Cerundolo, and P. R. Crocker. 2006. Characterization of Siglec-H as a novelendocytic receptor expressed on murine plasmacytoid dendritic cell precur-sors. Blood 107:3600–3608.

85. Zhang, J. Q., B. Biedermann, L. Nitschke, and P. R. Crocker. 2004. Themurine inhibitory receptor mSiglec-E is expressed broadly on cells of theinnate immune system whereas mSiglec-F is restricted to eosinophils. Eur.J. Immunol. 34:1175–1184.

86. Zhang, M., T. Angata, J. Y. Cho, M. Miller, D. H. Broide, and A. Varki. 2007.Defining the in vivo function of Siglec-F, a CD33-related Siglec expressed onmouse eosinophils. Blood 109:4280–4287.

87. Zhang, M., and A. Varki. 2004. Cell surface sialic acids do not affect primaryCD22 interactions with CD45 and surface IgM nor the rate of constitutiveCD22 endocytosis. Glycobiology 14:939–949.

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